Controlled Expression of Recombinant Proteins in Physcomitrella patens by a Conditional Heat-shock Promoter: a Tool for Plant Research and Biotechnology

Abstract

The ability to express tightly controlled amounts of endogenous and recombinant proteins in plant cells is an essential tool for research and biotechnology. Here, the inducibility of the soybean heat-shock Gmhsp17.3B promoter was addressed in the moss Physcomitrella patens, using b-glucuronidase (GUS) and an F-actin marker (GFP-talin) as reporter proteins. In stably transformed moss lines, Gmhsp17.3B-driven GUS expression was extremely low at 25°C. In contrast, a short non-damaging heat-treatment at 38°C rapidly induced reporter expression over three orders of magnitude, enabling GUS accumulation and the labelling of F-actin cytoskeleton in all cell types and tissues. Induction levels were tightly proportional to the temperature and duration of the heat treatment, allowing fine-tuning of protein expression. Repeated heating/cooling cycles led to the massive GUS accumulation, up to 2.3% of the total soluble proteins. The anti-inflammatory drug acetyl salicylic acid (ASA) and the membrane-fluidiser benzyl alcohol (BA) also induced GUS expression at 25°C, allowing the production of recombinant proteins without heat-treatment. The Gmhsp17.3B promoter thus provides a reliable versatile conditional promoter for the controlled expression of recombinant proteins in the moss P. patens.Abbreviations: ASA, acetyl salicylic acid; BA, benzyl alcohol; GFP, green fluorescent protein; GT, GFPtalin; GUS, b-glucuronidase; Fv/Fm, the maximal photochemical efficiency of PSII; MU, 4-methylumbelliferone; MUG, 4-methylumbelliferyl-b-D-glucuronide; PSII, photosystem II; SA, specific activity; sHSP, small heat-shock protein; TSP, total soluble proteins; Ubi, ubiquitin; X-Gluc, 5-bromo-4-chloro-3-indolyl-b-D-glucuronide

Membrane lipid composition affects plant heat sensing and modulates Ca2+-dependent heat shock response

“…2A and 3). This lower calcium transient correlated well with the consequent milder activation of the Hsp17.3B promoter observed in HSP-GUS line 28 grown at the same temperatures and submitted to 1 h heat shock at 35°C (Fig. 2B).…”

“…Physcomitrella patens (Gransden wild type) was grown on moss solid minimal medium overlaid with a cellophane disk as in Saidi et al 28 and maintained in a temperature-controlled chamber at indicated temperatures…”

“…Luminescence counts (CPS) were integrated every 3 s and, at the end of each monitoring, a discharging solution (0.1 M CaCl 2 , 10% ethanol, 2% Nonidet P40) was and durations. HSP-GUS and UBI-AEQ lines were previously described in Saidi et al 28 and Saidi et al 20 respectively. To perform heat shocks, protonemal tissues were transferred to liquid minimal medium using Costar multiwell plates (Corning Incorporated, Corning, NY) as detailed in Saidi et al 20 Fatty acid analysis.…”

“…These findings are in line with earlier reports that membrane fluidizers, such as benzyl alcohol, cause a HSR at non-inducing temperatures in plants and bacteria. 11,12,28 Thus, in Escherichia coli 19 and Synechocystis 29 preadaptation to different growth temperatures modulated the subsequent heat-induced expression levels of major classes of HSPs and affected thermotolerance.…”

The knock‐out of ARP3a gene affects F‐actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens

“…The consequence of loss of function of the ARP3a gene on the organization of the actin cytoskeleton was studied in the previously described HGT1 strain that carries a heat shock inducible GFP-talin reporter construct [Finka et al, 2007;Saidi et al, 2005]. The Hyg R HGT1 Fig.…”